Abstract

Lymphangioleiomyomatosis (LAM) is characterized by cystic lung destruction caused by LAM cells (smooth-muscle-like cells) that have mutations in the tumor suppressor genes tuberous sclerosis complex (TSC) 1 or 2 and have the capacity to metastasize. Since chemokines and their receptors function in chemotaxis of metastatic cells, we hypothesized that LAM cells may be recruited by chemokine(s) in the lung. Quantification of 25 chemokines in bronchoalveolar lavage fluid from LAM patients and healthy volunteers revealed that concentrations of CCL2, CXCL1, and CXCL5 were significantly higher in samples from LAM patients than those from healthy volunteers. In vitro, CCL2 or MCP-1 induced selective migration of cells, showing loss of heterozygosity of TSC2 from a heterogeneous population of cells grown from explanted LAM lungs. Additionally, the frequencies of single-nucleotide polymorphisms in the CCL2 gene promoter region differed significantly in LAM patients and healthy volunteers (p = 0.018), and one polymorphism was associated significantly more frequently with the decline of lung function. The presence (i.e., potential functionality) of chemokine receptors was evaluated using immunohistochemistry in lung sections from 30 LAM patients. Expression of chemokines and these receptors varied among LAM patients and differed from that seen in some cancers (e.g., breast cancer and melanoma cells). These observations are consistent with the notion that chemokines such as CCL2 may serve to determine mobility and specify the site of metastasis of the LAM cell.

Immunoreactive chemokine receptors in LAM nodules. Left, Immunoreactivity of patients (n = 30, except for CXCR6, n = 15; see supplemental Table IS) that reacted with Abs against the indicated receptors. CXCR6 was only determined on 15 patients. Asterisk (*) indicates the four differentially expressed receptors as shown in . Right, Representative images of LAM cells reacted with anti-CCR2 Abs that did not react with vascular and alveolar cells or with anti-CCR10 Ab that reacted with proliferative LAM areas, but not with vascular, normal alveolar, or lymphatic-like structures. Abs against CXCR2 and CXCR4 reacted with vascular endothelial cells and nuclei of type II pneumocytes as well as LAM cells, which reacted also with anti-CXCR1 Ab that reacted only weakly with vascular endothelial cells. There was no reaction with normal rabbit IgG.

Chemotactic migration and loss of TSC2 heterozygosity by cells grown from explanted lungs of patients with LAM. After incubation for 24 h without serum, the heterogeneous populations of cells were incubated in Boyden chambers where medium in lower chambers contained no additions (serum-free = control), 10% serum without or with 4 µM cytochalasin D (Cy), or CCL2, CCL27, or CCL28 (each 100 ng/ml). A, Amounts of cells (relative fluorescence units) that had migrated through filters in 18–20 h are reported as means ± SD of values from triplicate incubations in five experiments. B, Cells that had migrated through 24-well Boyden chambers were detached and DNA was extracted for analysis of the D16S3395 microsatellite marker on chromosome 16. In the top (control) chromatogram, the amplified alleles are from the DNA of a heterogeneous population; arrows indicate two alleles of TSC2. Two alleles were also seen in DNA from cells that migrated in response to FBS or in the presence of CCL28, but only one was observed among cells that responded to CCL2. Results were similar with cells from two patients.

Immunoreactivity of LAM cells to Abs against MCP-1/CCL2, CCL27, and CCL28. Upper, CCL2 immunoreactivity is seen in what appears to be extracellular matrix (not within LAM cells) of a LAM nodule as well as that of normal alveolar wall and outside of vascular structures. CCL27 and CCL28 immunoreactivity is seen in extracellular matrix area with only weak staining of LAM cells (magnification, ×100). Lower, Percentage of patients (n = 30) whose lung sections reacted with Abs against CCL2, CCL27, and CCL28.